Viral cell death protein and uses therefor

ABSTRACT

Viral proteins with sequence and functional similarity to the drosophila cell death protein known as Reaper are provided. Reaper plays an important role in the apoptotic pathway. Methods using the viral proteins and nucleotides encoding such proteins are described.

FIELD OF THE INVENTION

[0001] The present invention relates to a viral cell death gene and theprotein expressed thereby, and in particular, but not exclusively, tonucleotide sequences, expression vectors, cell lines, antibodies,screening methods, compounds, methods of production and methods oftreatment, related to them.

BACKGROUND OF THE INVENTION

[0002] Apoptosis is a form of programmed cell death, by which anorganism eliminates extraneous or harmful cells. Apoptosis plays a rolein normal development and homeostasis, as well as in diseases such ascancer and neurodegenerative diseases.

[0003] Apoptosis occurs via the activation of intrinsic cell-suicideprograms;

[0004] activation is regulated by many different signals, bothintracellular and extracellular. Various viral and metazoan apoptosisinducer genes have been identified; inhibitors of apoptosis (IAP)proteins have also been identified that act to suppress apoptosis.

[0005] At least three apoptotic activator proteins have been identifiedin Drosophila melaiiogaster: reaper (rpr), head involution defective(hid) and grim. The N-terminal sequences of these proteins are highlyconserved. Avdonin et al., Proc. Natl. Acad. Sci. USA 95:11703 (1998).Proteins that inhibit apoptosis (IAPs) have also been identified inDrosophila. Hay et al., Cell 83:1253 (1995); Wing et al., Cell DeathDiffer. 5:930 (1998).

[0006] The product of the reaper gene is required for programmed celldeath in Drosophila. Cell death induced by the reaper protein has beenshown to be blocked by the baculovirus p35 protein, a viral product thatinactivates proteases. White et al., Science 271:805 (1996).

[0007] The Drosophila Grim protein is also an activator of apoptosis,independent of Reaper. Expression of Grim RNA coincides with the onsetof programmed cell death during embryonic development, and ectopicinduction of grim has been show to trigger extensive apoptosis intransgenic animals and in cell culture. Similar to the Reaper protein,cell killing by grim can be blocked by coexpression of the viral p35product. The grim gene product has been postulated to function in aparallel cell death signalling regime that activates a common set ofdownstream apoptotic effectors. Chen et al., Genes Dev. 10:1773-1782(1996).

[0008] Viral infection of host cells, and replication therein, is oftenassociated with inhibition of apoptosis to enable viral replication andthe subsequent stimulation apoptosis of the host cells for viralparticle release. Certain viral gene products have been shown tospecifically inhibit or induce apoptosis. However, many virusesadditionally encode proteins that inhibit apoptosis, prolonging thesurvival of infected cells and thereby aiding viral replication or viralpersistence in the host.

SUMMARY OF THE INVENTION

[0009] A first aspect of the present invention is a method of screeningcompounds for the ability to modulate the apoptotic effects of viralreaper proteins on cells. The protein may consist of or comprise anamino acid sequence selected from SEQ ID NOs:2-17.

[0010] According to another aspect of the invention there is provided amethod of using nucleotide sequences encoding viral reaper proteins or avariant thereof, or a nucleotide sequences which are complementarythereto. Preferably the nucleotide sequence is a cDNA sequence.Particularly preferably the nucleotide sequence is selected from SEQ IDNOs:2-17.

[0011] According to another aspect of the invention there is provided amethod of treating cells using viral reaper proteins, to affect thecell's apoptotic mechanism.

[0012] According to another aspect of the invention, there is provided amethod of using expression vectors comprising a nucleic acid sequence asreferred to above that is capable of expressing a viral reaper protein.

[0013] According to another aspect of the invention there is provided amethod of treatment or prophylaxis of a disorder that is responsive tomodulation of apoptosis by a viral reaper protein modulator, the methodcomprising administering to a subject in need thereof an effectiveamount of a viral reaper protein modulating compound. Preferably thedisorder is selected from viral infections in mammalian subjects.

[0014] According to a further aspect of the invention there is provideduse of a compound that modulates viral reaper protein activity in amethod of formulating a medicament for treatment or prophylaxis of adisorder that is responsive to modulation of viral reaper proteinactivity, in a subject in need of such treatment. Preferably thedisorder is selected from viral infections in mammalian subjects.

BRIEF DESCRIPTION OF THE FIGURES

[0015] The present invention will be further described by way of exampleand with reference to the following figures:

[0016]FIG. 1 shows the amino acid sequences of the nonstructural (NSs)viral proteins from fifteen different virus, aligned with the amino acidsequence of the complete Reaper protein (SEQ ID NO: 1) and a portion ofthe Grim protein from Drosophila inelanogaster (SEQ ID NO:18). Amajority sequence for the viral reaper protein is provided as SEQ IDNO:2.

[0017]FIG. 2 shows Western blot results indicating binding of variousreaper proteins to Xenopus scythe protein (Example 4). RPR1 and RPR2indicate preparations of Drosophila Reaper, SA indicates San Angelovirus reaper, and CE indicates California Encephalitis virus reaper;GST1 and GST2 are preparations of glutathione-S-transferase used ascontrols.

[0018]FIG. 3 graphs the results of a colorimetric assay to detectcaspase activation in Xenopus cell-free extracts with added Drosophilareaper (Rpr 4/5, Rpr 12/00, Rpr 12/20), San Angelo virus reaper (SA),and California Encephalitis virus reaper (CE). Glutathione-S-transferase(GST) was used as a control. The Y-axis shows absorbance at 405 nm; theX-axis =time in hours.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Viral proteins with sequence similarity to the Drosophilamelanogaster Reaper protein have been identified, and shown to becapable of activating caspases in vertebrate cell-based assay. Cellulardeath by apoptosis is, with only some exceptions, executed by the familyof proteases known as caspases. The caspases are synthesized as inactivezymogens and activated during apopototic pathways. (Chinnaiyan andDixit, Curr. Biol. 6:555 (1996); Muzio et al, J. Biol. Chem. 273:2926(1998); Yang et al., Mol. Cell. 1:319 (1998); Zou et al., Cell 90:405(1997)). Caspase activation (and thus ultimately apoptosis) can beblocked by the IAP (inhibitor of apoptosis) proteins and anti-apoptoticmembers of the Bcl-2 family (Adams and Cory Science 281:1322 (1998);Deveraux and Reed, Genes Dev. 13:239 (1999)).

[0020] The present viral genes and their expression products provideuseful screening assays for the identification and development of novelpharmaceutical agents, including agonists and antagonists of the viralreaper proteins, and compounds able to enhance or inhibit caspaseactivation and/or cellular apoptosis; such compunds may be used in thetreatment and/or prophylaxis of disorders such as cancer and viralinfections. In particular, the present methods are useful in identifyingcompounds that enhance or inhibit apoptosis due to caspase activation.

[0021] Accordingly, it is an object of the present invention to providemethods of using isolated viral reaper proteins. Other objects of thepresent invention will become apparent from the following detaileddescription thereof.

[0022] The present inventors identified viral proteins with sequencesimilarity to the D. melanogaster Reaper protein. More particularly, thepresent inventors determined that the non-structural protein (NSs) ofviruses of the genus Bunyavirus exhibited sequence similarity to theDrosophila Reaper protein.

[0023] Viruses of the genus Bunyavirus (family Bunyaviridae) are singlestranded RNA negative-strand viruses that infect vertebrates. There are18 antigenic groups of the genus Bunyavirus (at least 161 viruses) andseveral additional ungrouped viruses.

[0024] The California encephalitis virus, LaCrosse virus, San Angelovirus, Snowshoe Hare virus, Jerry Slough virus, Jamestown Canyon virus,Keystone Virus, Melao virus, Trivittatus virus, Morro Bay virus, Inkoovirus, Serra do Navio virus, South River virus, Lumbo virus, and Tahynavirus are each members of the genus Bunyavirus. The genome consists of alarge (L) RNA, a medium (M) RNA, and a small (S) RNA. The nonstructuralprotein (NSs protein) of these viruses is encoded by the small (S) RNA,and is induced in virus-infected cells (Fuller and Bishop (1982) J.Virol. 41, 643-648). The NSs protein of each of these viruses was foundby the present inventors to have sequence similarity to the DrosophilaReaper protein (FIG. 1).

[0025] A nucleotide sequence encoding D. melanogaster Reaper protein isgiven at GenBank Accession No. L31631; the protein sequence is providedat Acc. No. AAA18983. The amino acid sequence of the Drosophila celldeath protein GRIM is provided at GenBank Ace. No. AAC47727.

[0026] The nucleotide sequence encoding San Angelo virus (PrototypeVR723) nucleocapsid and non-structural proteins is provided at GenBankAccession U47139. The amino acid sequence of San Angelo virus NSsprotein is provided at Ace No. AAC5335.

[0027] The amino acid sequence of a NSs of the Snowshoe Hare virus isprovided at Accession No. P03514; the nucleotide sequence of a small (S)viral RNA species of snowshoe hare virus is provided at Ace. No. J02390.See also Bishop et al., Nucleic Acids Res. 10 (12), 3703-3713 (1982).

[0028] The nucleotide sequence of a La Crosse virus small RNA segment isprovided at GenBank Accession No. K00108. The amino acid sequence for aLa Crosse Virus non structural protein can be found at Accession No.AAA42780.1

[0029] The nucleotide sequence for a California Encephalitis virus smallRNA segment, including the nucleocapsid and NSs protein genes, isprovided at GenBank Accession No U12797; the amino acid sequence of theviral NSs protein can be found at Acc. No. AAC54056.1. See also Bowen,M. D., et al. J. Gen. Virol. 76: 559-572 (1995).

[0030] The nucleotide sequence encoding Jerry Slough Virus NSs proteinis provided at GenBank Accession No. U12798; the amino acid sequence ofthe viral NSs protein can be found at Acc. No. AAC54048.

[0031] The nucleotide sequence encoding Jamestown Canyon Virus NSsprotein is provided at GenBank Accession No. U12796; the amino acidsequence of the viral NSs protein can be found at Acc. No. AAC54044.

[0032] The nucleotide sequence encoding Keystone Virus NSs protein isprovided at GenBank Accession No. U12801; the amino acid sequence of theviral NSs protein can be found at Acc. No. 54054.

[0033] The nucleotide sequence encoding Melao Virus NSs protein isprovided at GenBank Accession No. U12802; the amino acid sequence of theviral NSs protein can be found at Acc. No. AAB60560.

[0034] The nucleotide sequence encoding Trivittatus Virus NSs protein isprovided at GenBank Accession No. U12803; the amino acid sequence of theviral NSs protein can be found at Acc. No. AAB60562.

[0035] The nucleotide sequence encoding Morro Bay Virus NSs protein isprovided at GenBank Accession No. U31989; the amino acid sequence of theviral NSs protein can be found at Acc. No. AAC55125.

[0036] The nucleotide sequence encoding Inkoo Virus NSs protein isprovided at GenBank Accession No. U47138; the amino acid sequence of theviral NSs protein can be found at Acc. No. AAC55333.

[0037] The nucleotide sequence encoding Serra do Navio Virus NSs proteinis provided at GenBank Accession No. U47140; the amino acid sequence ofthe viral NSs protein can be found at Acc. No. AAC55337.

[0038] The nucleotide sequence encoding South River Virus NSs protein isprovided at GenBank Accession No. U47141; the amino acid sequence of theviral NSs protein can be found at Acc. No. AAC55339.

[0039] The nucleotide sequence encoding Lumbo Virus NSs protein isprovided at GenBank Accession No. X73468; the amino acid sequence of theviral NSs protein can be found at Acc. No. CAA51852.

[0040] The nucleotide sequence encoding Tahyna Virus NSs protein isprovided at GenBank Accession No. Z68497; the amino acid sequence of theviral NSs protein can be found at Ace. No. Z68497.

[0041] The present viral Reaper proteins act as apoptotic modulatingproteins, affecting apoptosis in host cells. Discovery of these viralproteins provides methods to screen compounds for the ability to blockor enhance the viral reaper function, and the use of compoundsidentified thereby to modulate apoptosis. Apoptosis may also bemodulated by the administration of viral reaper proteins (or afunctional variant or fragment thereof) to a cell. Administration mayutilize isolated nucleic acid molecules encoding the viral reaperproteins, vectors containing such molecules, and host cells transfectedwith the same. The host cells may be any cell suitable for cultivationof the virus, as is known in the art. More preferred are vertebratecells, including mammalian cells.

[0042] The present proteins (and functional variants and fragmentsthereof) and nucleotides expressing the same, are useful in severalsettings. Where a virus is being cultivated, either in cell culture orin vivo, the presently identified viral reaper proteins (and/or thecompounds discovered in the screening assays described above) may beused to modulate apoptosis of infected host cells, by administering theprotein or compounds to the cells. Inhibiting apoptosis of the host cellis useful where further replication of the virus within the host cellcan be attained and is desirable. Inducing apoptosis of the host cell isuseful where infection of additional cells is desirable, in harvestingvirus, or where it is necessary and desirable to terminate survival ofthe host cell in order to halt further viral replication/propagation.

[0043] Additionally, the present proteins and/or compounds discoveredusing the present screening assays, may be used in the treatment ofcertain conditions involving aberrant apoptosis of a subject's cells.Viral reaper proteins and/or such compounds may be administered to apreselected population of cells in a subject, to modulate apoptosis ofthose cells. Such a method is useful in conditions where the normalapoptotic mechanism of the subject's cells is altered, e.g., in cancerand neoplastic growths. Such a method is also useful in treating viralinfections.

[0044] The present invention also provides a method to screen compoundsfor the ability to inhibit or enhance the function of the viral reaperproteins (e.g., to act as antagonists or agonists of the viral reaperprotein function). Such compounds will be useful in the above-describedmethods. Such compounds may be administered alone, to act uponendogenous viral reaper proteins produced within an infected host cell;or they may be administered to a cell in conjunction with exogenousviral reaper protein where needed (e.g., where the compound enhances theeffects of the exogenous viral reaper protein).

[0045] As used herein, “sequence similarity” of proteins refers to thesimilarity of the amino acid sequence between two proteins. Variouscomputer programs are commercially available that determine sequencesimilarity, and are known to those skilled in the art. The degree ofsequence similarity takes into consideration both amino acid residuesthat are identical, as well as those that are conservative amino acidsubstitutions (as is known in the art).

[0046] “Sequence similarity” as used in the present specification andclaims refers to DNA sequences, (or RNA sequences; or amino acidsequences) that have only slight and non-consequential sequencevariations between them. In this regard, ‘slight and non-consequentialsequence variations’ mean that the sequences will be functionallyequivalent. Functionally equivalent sequences will function insubstantially the same manner to produce substantially the same results.

[0047] As used herein, two amino acid sequence that have substantial“sequence similarity” are those having at least about 50% or 55%sequence similarity, and preferably at least about 60%, 65%, 70%, 75%,80%, 85%, or even about 90% or 95% similarity. Changes in the amino acidsequence of peptides can be guided by known similarities among aminoacids and other molecules or substituents in physical features such ascharge density, hydrophobicity, hydrophilicity, size and configuration,etc. For example, the amino acid Thr may be replaced by Ser and viceversa, and Leu may be replaced by lie and vice versa.

[0048] The peptides of the present invention include not only naturalamino acid sequences, but also peptides which are analogs, chemicalderivatives, or salts thereof. The term “analog” or “conservativevariation” refers to any polypeptide having a substantially identicalamino acid sequence to the peptides identified herein, and in which oneor more amino acids have been substituted with chemically similar aminoacids. For example, a polar amino acid such as glycine or serine may besubstituted for another polar amino acid; a basic amino acid may besubstituted for another basic amino acid, or an acidic amino acid may besubstituted for another acidic amino acid; or a non-polar amino acid maybe substituted for another non-polar amino acid. There term “analog” or“conservative variation” as used herein also refers to a peptide whichhas had one or more amino acids deleted or added to a polypeptide of thepresent invention, but which retains a substantial sequence similarity(at least about 85% sequence similarity, and preferably at least 90%,95%, 98% or even 99% sequence similarity), where the peptide retains theviral reaper protein function or generates an antagonistic viral reaperfunction.

[0049] Given the amino acid sequence of a particular viral protein, anucleotide sequence encoding the protein can be readily determined, anda nucleotide molecule encoding the protein can be prepared.

[0050] The present invention also encompasses the use of nucleotidemolecules that encode viral reaper proteins. Examples of such nucleotidesequences are below. Due to the degeneracy of the genetic code, oneskilled in the art will be able to readily devise alternative nucleotidesequences that encode a given protein, where the amino acid sequence ofthe protein is known. nucleotide sequence encoding Drosophilamelanogaster Reaper Protein L31631 (SEQ ID NO:19) atggcagtgg cattctacatacccgatcag gcgactctgt tgcgggaggc ggagcagaag 60 gagcagcaga ttctccgcttgcgggagtca cagtggagat tcctggccac cgtcgtcctg 120 gaaaccctgc gccagtacacttcatgtcat ccgaagaccg gaagaaagtc cggcaaatat 180 cgcaagccat cgcaatganucleotide sequence encoding San Angico Virus NSs protein U47139 (SEQ IDNO:20) atgatgtcgc atcaaccggt gcaaatggat ttgatcctga tgcagggtat ctggcattct60 gtgttaaaca tggggagtcg atcagtttgt cttcagttag gatcttcttc ctcaatgccg 120caaaagccaa agctgctctc tcgcgtaaac cagagaggaa agcaaatcct aaatttggcg 180agtggcaggt ggagattgtc aataatcatt ttcctggaaa caggaacaat ccaattgaca 240acctcgatct taccatccac agattgtctg gatacctggc tagatqggtt ctag 294nucleotide sequence encoding Snowshoe Hare Virus UP03514 (SEQ ID NO:21atgatgtcgc atcaacaggt gcaaatggat ttgatcctga tgcagggtat atggcattct 60gtgttaaata tgcagaatca gtcaatcttg ctgcagttag gatcttcttc ctcaatgccg 120caaaggccaa ggctgctctc tcgcgtaagc cagagaggaa ggcaaatcct aaatttggag 180agtggcaggt ggaggttgtc aataatcatt ttcctggaaa caggaacaat ccaattaaca 240gcgacgatct taccatccac agattgtcag gatatttag nucleotide sequence encodingLaCrosse Virus NSs protein K00108 (SEQ ID NO:22) atgatgtcgc atcaacaggtgcaaatggat ttgatcctga tgcagggtat atggacttct 60 gtgttaaaaa tgcagaatcactcaaccttg ctgcagttag gatcttcttc ctcaatgctg 120 caaaggccaa ggctgctctctcgcgtaagc cagagaggaa ggctaaccct aaatttggag 180 agtggcaggt ggaggttatcaataatcatt ttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccatccacagattatctg ggtatttag nucleotide sequence encoding Californiaencephalitis NSs protein U12797 (SEQ ID NO:23) atgatgtcgc atccacaggtgcaaatggat ttgatcctga tgcagggtat gtggacttct 60 gtgctaaaca tggggaatcaattaaccttg ctgcagttag gatcttcttc ctcaatgccg 120 caaaggccaa ggctgctctctcgcgtaagc cagagaggaa agctaatcct aaatttggcg 180 agtggcaggt ggaggttgtcaataatcatt ttccagcaaa caggaacaat ccaattggta 240 acaacgatct taccatccaccgcatctcag gataccttgc cagatgggtc ctag 294 nucleotide sequence encodingJerry slough virus NSs protein U12798 (SEQ ID NO:24) atgatgtcgcatccacaggt gcaaatggat ttgatccaga tgcagggttt gtggcattta 60 tggctgaccacggagagtct atcaatctgt cagccgttag gatcttcttc cttaatgcag 120 caaaagccaaagctgctctc gctcgtaaac cggagcggaa agctactcct aagtttggag 180 agtggcaggtggagatcatc aataatcatt ttcctggaaa caggaacaac ccaattggta 240 acaacgatcttaccatccat aggctttcag gatatctag 279 Nucleotide sequence encodingJamestown Canyon virus NSs protein U12796 (SEQ ID NO:25) atgatgtcgcatccacaggt gcaaatgqat ttgatccaqa tgcagggttt gtggcattta 60 tggctgaccacggagagtct atcaatctgt cagccgttag gatcttcttc cttaatgcag 120 caaaagccaaagctgctctc gctcgtaaac cggagcggaa agctactcct aagtttggag 180 agtggcagqtggagatcgtc aataatcatt tttctggaaa caggaacaac ccaattggta 240 acaacqatcttaccatccat aggctttcag gatatctag 279 Nucleotide sequence encodingKeystone Virus NSs protein U12801 (SEQ ID NO:26) atgatgtcgc atccacaggtgcaaatggat ttgatcctga tgcagggtat gtggcattta 60 tggctaacca tggggagtcgatcagtctgt caaccgttag gatcttcttc cttaatgccg 120 caaaagccaa agctgctctcactcgtaagc cggagcggaa ggctacacct aagtttggag 180 agtggcaggt ggagatcgtcaataatcatt ttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccttgcaccggatttcag gatatctag 279 Nucleotide sequence encoding Melao Virus NSsprotein U12802 (SEQ ID NO:27) atgatgtcgc atcaacaggt gcaaatggatttgatccaga tgcagggtat ctggcattta 60 caattgcgca tggggaagct atcaatttgtcagccgttag gatcttcttc cttaatgccg 120 caaaagccaa agctgctctc tctcgtaaaccggagaggaa agctactcct aaatttggag 180 actggcaggt ggaaattgtc aacaatcattttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccatccat cggctttcaggatatcttgc cagatgggtg ctag 294 Nucleotide sequence encoding TrivittatusVirus NSs protein U12803 (SEQ ID NO:28) atgatgctcc atcaacaggt gcaaacggatttgatcccga tgcagggtat gtggcattta 60 ttgctgcaca tgccggatcg tacgatctttctgctgttag gatcttcttc ctcaatgctg 120 ccaaggccaa gaatgctctc tcgagaaaaccagaggggaa ggttagtatt aaatttggcg 180 agtggtcggt ggaggtggtc aataatcattttcctggcaa caggaacaat ccaattggta 240 acaacgatct taccatccac agaatttcaggctatctcgc aagatgggtt ctag 294 Nucleotide sequence encoding Morro BayVirus NSs protein U31989 (SEQ ID NO:29) atgatgtcgc atccacaggt gcaaatggatttgatcctga tgcagggtat gtggacttct 60 gtgctaaaca tggggaatcg attaaccttgctgcagttag gatcttcttc ctcaatgccg 120 caaaagccaa ggctgctctc tcgcgtaagccagagaggaa agctaatcct aaatttggcg 180 agtggcaggt ggagattgtc aataatcattttccagcaaa caggaacaat ccaattggta 240 acaacgatct taccatccac cgcatctcaggataccttgc cagatgggtc ctag 294 Nucleotide sequence encoding Inkoo VirusNSs protein U47138 (SEQ ID NO:30) atgatgtcgc atccacaggt gcaaatggatttgatccaga tgcagggttt gtggcattta 60 tggctgacca tggagaatct attaatttggcagccgttag gatcttcttc cttaatgcag 120 caaaagccaa agctgctctc gctcgtaaaccggagcggaa agctactcct aaatttggag 180 agtggcaggt ggagattgtc aataatcattttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccatccac cggctttctggatacttag 279 Nucleotide sequence encoding Serra do Navio Virus NSsprotein U47140 (SEQ ID NO:31) atgatgtcgc atcaaccggt gcaaatggatttgatccaga tgcagggttt gtggcattta 60 tggctggtca tggggagtcg atcaatcttacagccgttag aatcttcttc cttaatgccg 120 caaaagccaa agctgctctc tctcgcaagccggagaggaa agctactcct aagtttggag 180 actggcaggt ggagattgtc aataatcattttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccatccac agaatttcaggatatttag 279 Nucleotide sequence encoding South River Virus NSs proteinU47141 (SEQ ID NO:32) atgatgtcgc atccacaggt gcaaatggat ttgatccagatgcagggttt gtggcattta 60 tggctgacca tggagaatct atcaatctgt cagccgttaggatcttcttc cttaatgcag 120 caaaagccaa agctgctctc gctcgtaaac cggagcggaagqctaatcct aaatttggag 180 agtggcaggt ggagattgtc aataatcatt ttcctggaaacaggaacaac ccaattggta 240 acaacgatct taccatccac cggctttctg gatatttag 279Nucleotide sequence encoding Lumbo Virus NSs protein X73468 (SEQ IDNO:33) atgatgtcgc atccaccggt gcaaatggat ttgatcctga tgcagggtat gtggactttt60 gtgttaaaca tggagaatca atcaatctcc attccgttag gatctttttc cttaatgccg 120ctaaggccaa ggctgctctc gctcgtaagc cggagaggaa ggctagtcct aaatttggag 180agtggcaggt ggagatcgtc aataatcatt ttcctggaaa caggaacaac ccaattgata 240acaacgatct taccatccac cggctgtcag ggtatctggc tagatgggtg ttag 294Nucleotide sequence encoding Tahyna Virus NSs protein Z68497 (SEQ IDNO:34) atgatgtcgc atccaccggt gcaaatggat ttgatcctga tgcagggtat gtggacttct60 gtattaaaca tggggaagca attaatctcc attccgttag gatcttcttc cttaatgccg 120caaaagccaa agctgctctc gctcgtaagc cggagaggaa ggctagtcct aaatttggag 180agtggcaggt ggaggtcgtc aattatcatt ttcctggaaa caggaacaac ccaattgata 240acaacgatct taccatccac cggctgtacg ggtatttggc tagatgggtg ctag 294

[0051] Nucleotide sequences that have substantial sequence similarity tothe nucleotide sequences disclosed herein, and that encode functionalviral reaper proteins, are suitable for use in the methods of thepresent invention.

[0052] Thus, use of nucleotides that hybridize to nucleotide moleculesencoding the viral reaper proteins of SEQ ID NOs:2-17 are also an aspectof this invention. Conditions which will permit other nucleotidemolecules encoding viral reaper proteins to hybridize to the nucleotidesequences disclosed herein can be determined in accordance with knowntechniques. For example, hybridization of such sequences may be carriedout under conditions of reduced stringency, medium stringency, or evenhigh stringency conditions (e.g., conditions represented by a washstringency of 35-40% formamide with 5× Denhardt's solution, 0.5% SDS and1× SSPE at 37° C.; conditions represented by a wash stringency of 40-45%formamide with 5× Denhardt's solution, 0.5% SDS, and 1× SSPE at 42° C.;and conditions represented by a wash stringency of 50% formamide with 5×Denhardt's solution, 0.5% SDS and 1× SSPE at 42° C., respectively, to anucleotide molecule encoding a viral reaper protein as disclosed hereinin a standard hybridization assay. See J. Sambrook et al., MolecularCloning: A Laboratory Manual (2d ed. 1989)). In general, sequencesencoding functional viral reaper proteins that hybridize to thenucleotide sequences disclosed herein will have at least 30% sequencesimilarity, 50% sequence similarity, 75% sequence similarity, and even95% sequence similarity or more with the nucleotide sequences encodingviral reaper proteins disclosed herein.

[0053] Throughout the present specification and the accompanying claimsthe words “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

[0054] As referred to above, the present invention relates to methods ofusing isolated viral reaper proteins or isolated nucleotide moleculesencoding viral reaper proteins. In the context of this invention theterm “isolated” is intended to convey that the protein is not in itsnative state, insofar as it has been purified at least to some extent orhas been synthetically produced, for example by recombinant methods. Theterm “isolated” therefore includes the possibility of the protein beingin combination with other biological or non-biological material, such ascells, suspensions of cells or cell fragments, proteins, peptides,expression vectors, organic or inorganic solvents, or other materialswhere appropriate, but excludes the situation where the protein ornucleic acid molecule is in a state as found in nature.

[0055] As used herein, the term “viral reaper protein” comprisesproteins having an amino acid sequence identical to that of a proteinnaturally expressed by a virus, and having the ability to induce caspaseactivation in a vertebrate cell. One method of assaying a protein forthe ability to induce caspase activation in a vertebrate cell (Xenopusoocytes) is provided in the Examples section herein.

[0056] Routine methods, as further explained in the subsequentexperimental section, can be employed to purify and/or synthesize theproteins according to the invention. Such methods are well understood bypersons skilled in the art, and include techniques such as thosedisclosed in Sambrook J., Fritsch E. F. and Maniatis T., MolecularCloning: a Laboratory Manual; 2^(nd) Edition; CSH Laboratory Press(1989), the disclosure of which is included herein in its entirety byway of reference.

[0057] The term “variant” as used herein refers to peptides or proteinswhich retain the same essential characteristics (functional andstructural) of the viral Reaper proteins for which sequence informationis provided herein; such variants are intended to be included within thescope of the invention. For example, other peptides or proteins withgreater than about 50%, 55%, 60% or 65%, preferably at least 75% andparticularly preferably at least 80%, 90% or 95% sequence similaritywith the sequences provided, are considered as variants of the proteins.Such variants may include the deletion, modification or addition ofsingle amino acids or groups of amino acids within the protein sequence,as long as the peptide maintains the basic biological functionality of aviral Reaper protein. This biological functionality can be assessed byone skilled in the art using methods that are known in the art.

[0058] The term “protein” as used herein is also intended to includewithin its meaning shorter peptide or polypeptide sequences as well ascomplete proteins. For example therefore a peptide of only perhaps 15amino acids in length is considered to fall within the scope of theinvention as long as it demonstrates the basic biological functionalityof a viral Reaper protein as described herein. In particular, but notexclusively, this aspect of the invention encompasses the situation whenthe protein is a fragment of the complete protein sequence and mayrepresent a ligand binding region.

[0059] The invention also includes methods of using isolated nucleotidesequences that encode viral Reaper proteins or variants thereof, as wellas isolated nucleotide sequences which are complementary thereto. Thenucleotide sequence may be RNA or DNA including genomic DNA, syntheticDNA or cDNA. Preferably the nucleotide sequence is a DNA sequence andmost preferably, a cDNA sequence. Nucleotide sequence information isprovided herein for certain viral reapers. Such nucleotides can beisolated from virally infected cells or synthesised according to methodswell known in the art, as described by way of example in Sambrook J,Fritsch E. F. and Maniatis T; Molecular Cloning: a Laboratory Manual;2^(nd) Edition; CSH Laboratory Press (1989), the disclosure of which isincluded herein in its entirety by reference. The nucleotide moleculesaccording to the invention have utility in production of the proteinsaccording to the invention, which may take place in vitro, in vivo or exvivo. The nucleotides may be involved in recombinant protein synthesisor indeed as therapeutic agents in their own right, utilised in genetherapy techniques. Nucleotides complementary to those encoding viralReaper proteins of the present invention, or antisense sequences, mayalso be used in therapy, such as in strategies for down regulation ofexpression of the proteins of the invention.

[0060] The present invention also includes methods of using expressionvectors that comprise nucleotide sequences encoding viral Reaperproteins or variants thereof. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for protein expression. Suitable vectors would beapparent to persons skilled in the art. By way of further example inthis regard we refer to Sambrook J, Fritsch E. F. and Maniatis T;Molecular Cloning: a Laboratory Manual; 2^(nd) Edition; CSH LaboratoryPress (1989), the disclosure of which is included herein in itsentirety.

[0061] The invention also includes methods of using cell lines that havebeen modified to express viral reaper proteins. Such cell lines includetransient, or preferably stable higher eukaryotic cell lines, such asvertebrate cells, mammalian cells or insect cells; lower eukaryoticcells, such as yeast; or prokaryotic cells such as bacterial cells.Preferred are bacterial, insect and vertebrate cells. As used herein,cells that have been “modified” to express viral reaper proteins arethose that contain isolated nucleic acid molecules coding for a viralreaper protein, and excludes cells infected with a virus expressingviral reaper protein from a non-isolated nucleic acid (e.g., a naturallyoccurring virus).

[0062] According to another aspect, the present invention also relatesto antibodies (either polyclonal or preferably monoclonal antibodies)which have been raised by standard techniques and are specific for theproteins or variants thereof according to the invention. Such antibodiescould for example, be useful in purification, isolation or screeninginvolving immuno precipitation techniques and may be used as tools tofurther elucidate the protein function, or indeed as therapeutic agentsin their own right. Antibodies may also be raised against specificepitopes of the proteins according to the invention.

[0063] A further aspect of the present invention is the use of theproteins according to the invention in screening methods designed toidentify those compounds that act as ligands for viral Reaper proteins,and/or that modulate viral Reaper protein activity. In general terms,such screening methods will involve contacting the protein concerned, orcell modified to express the protein concerned, with a test compound andthen detecting any enhancement or inhibition of protein activity thatresults (compared to the activity that would occur in the absence of thetest compound). The present invention also includes within its scopethose compounds that are identified as possessing useful viral Reaperprotein modulation activity, by the screening methods referred to above,and use of such compounds. The screening methods comprehended by theinvention are generally well known to persons skilled in the art.

[0064] Another aspect of the present invention is the use of compoundsthat have been identified by screening techniques referred to above, orthe use of an isolated viral reaper protein, in the treatment orprophylaxis of disorders which are responsive to modulation of viralReaper protein activity, in a subject in need of such treatment.Preferably such subjects are vertebrates, and more preferably, mammals.The term “modulation”, as used herein, refers to both agonism(enhancement) and antagonism (inhibition) of an activity. Thusmodulation may consist of an increase (or a decrease or time delay) incaspase activation or cellular apoptosis, in response to administrationof a viral reaper protein (or a compound identified by the screeningmethods described herein). The change in activity (increase or decrease)is compared to that which would occur in the absence of the added viralreaper protein (or compound). Such increase (or decrease) may bemeasured by any suitable method as is known to those skilled in the art,e.g., detecting caspase activation over time or counting the number ofapoptotic cells in a population over a fixed time period. The modulationactivity may be due to direct binding of a compound to the viral reaperprotein, or due to the effects of the compound on a downstream elementof the apoptotic process that is modulated by the viral Reaper protein.Disorders that are responsive to modulation of cellular apoptosis arethose in which the signs, symptoms and/or pathological changesassociated with the disorder can be diminished or improved by altering(increasing, decreasing or delaying) cellular apoptosis.

[0065] Methods of screening compounds comprise contacting a viral reaperprotein with a test compound, or administering a test compound to a cellthat contains or expresses a viral reaper protein. By contacting it ismeant that the test compound and viral reaper protein are in suchproximity that they are able to biologically interact. Administration ofa compound to a cell refers to the placement of the compound within thecell interior, either by direct administration or by cellular uptake.

[0066] A particular method of screening a compound to determine whetherthe compound enhances or inhibits caspase activation utilizes vertebratecell extracts (cell-free preparations) which are known to exhibitdetectable caspase activation when functional viral reaper is added tothe cell extract. As used herein, an increase in (or enhancement of)caspase activation includes an increase in total caspase activity, afaster rate of caspase activity, and/or a decreased time until caspaseactivity is detected, as well as other measures that will be apparent tothose skilled in the art. As used herein, a decrease in (or inhibitionof) caspase activation includes a decrease in total caspase activity, aslowed rate of caspase activity, and/or an increase in the time untilcaspase activity is first detected; as well as other measures that willbe apparent to those skilled in the art. Other indicators of activationof the apoptotic pathway are known in the art, e.g., mitochondrialcytochrome c release, fragmentation of nuclei added to a cell extractpreparation. The change in caspase activation (or other indicators ofapoptosis) is compared between preparations that contain the testcompound and control preparations that do not; however, such comparisonsneed not be a side-by-side comparison, where a control has previouslybeen tested and has provided data for comparison.

[0067] As used herein, a compound with “viral reaper modulatingactivity” is one that is capable of enhancing or inhibiting viral reaperinduced apoptosis or caspase activation. As used herein, apoptosisinduced by a viral reaper protein is that caused by activation of theapoptotic pathway by the viral reaper.

[0068] Some specific examples of disorders that may be treated orprevented by administration of compounds identified in the screeningtechniques according to the present invention are viral infections anddisorders of apoptosis (such as cancer and neoplastic growths). Mentionof such disorders is by way of example only, and is not intended to belimiting on the scope of the invention as described.

[0069] The compounds identified according to the screening methodsoutlined above may be formulated with standard pharmaceuticallyacceptable carriers and/or excipients as is routine in thepharmaceutical art, and as fully described in Remmington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th Ed,1985, the disclosure of which is included herein in its entirety by wayof reference.

[0070] The compounds may be administered via enteral or parenteralroutes such as via oral, buccal, anal, pulmonary, intravenous,intraarterial, intramuscular, intraperitoneal, topical or otherappropriate administration routes. Appropriate administration routes,effective amounts, and dosing regimes may be determined by one skilledin the art using methods known in the art, e.g., dosage/responsestudies.

[0071] The present invention will now be further described by way ofexample.

EXAMPLES Example 1 Identification of Viral Reaper Proteins

[0072] Using Advanced Blast, Psi Blast, Edit Seq and Meg Align programs,15 nonstructural viral proteins (or the in silico translated viral DNAfor these proteins) were unexpectedly found to have sequence similarityto the Drosophila melanogaster Reaper protein.

[0073] The 15 viral proteins (or in silico translated DNAs) investigatedwere the non-structural proteins (NSs) of San Angelo virus (NSs) (SEQ IDNO:3); Snow Shoe Hare virus (SEQ ID NO:4); La Crosse virus (SEQ IDNO:5), California encephalitis virus (SEQ ID NO:6), Jerry Slough virus(SEQ ID NO:7), Jamestown Canyon virus (SEQ ID NO:8), Keystone virus (SEQID NO:9), Melao virus (SEQ ID NO:10), Trivittatus virus (SEQ ID NO:11),Morro Bay virus (SEQ ID NO:12), Inkoo virus (SEQ ID NO:13), Serra doNavio virus (SEQ ID NO:14), South River virus (SEQ ID NO:15), Lumbovirus (SEQ ID NO:16), and Tahyna virus (SEQ ID NO:17). The amino acidsequences of these proteins were compared to that of the D. melanogasterReaper protein (SEQ ID NO:1) and a portion of the D. melanogaster Grimprotein (SEQ ID NO:18) Results are shown in FIG. 1.

[0074] It was found that 59 amino acids of the 65 amino acid drosophilareaper protein aligned with 61 amino acids of the ˜92 amino acid viralreaper protein (or in silico translated DNA) with 58% similarity and 29%identity. These points of alignment therefore provide for a consensusreaper sequence cross-species.

[0075] A majority sequence was prepared (SEQ ID NO:2).

Example 2 Function of Viral Reaper Proteins

[0076] Viral reaper activity is detected utilizing extracts preparedfrom Xenopus eggs as described in Thress, et al, 1998, The EMBO Journal17:6135-6143. Briefly, an egg extract prepared as described, is treatedwith 10 to 1000 nanograms of viral reaper protein or a glutathione-Stransferase (GST) fusion protein with viral reaper. Control extracts(without viral reaper) are also prepared. After incubation of themixture, caspase enzyme activity is measured with the substrate DEVD-pNAby following the increase in absorbance at 405 nanometers. Proteins suchas reaper that are apoptotic activators cause an acceleration of theproduction of caspase activity (compared to that seen in controls).

[0077] The presence of apoptotic inhibitors slows or reduces caspaseactivation in extracts where reaper is incubated with the extract(compared to extracts incubated with reaper that do not contain theapoptotic inhibitor).

[0078] Ectopic expression of Drosophila Reaper has also been shown toinduce caspase activation and apoptosis in mammalian cells such as humanMCF7 breast carcinoma cells (McCarthy & Dixit, J. Biol. Chem. 273:24009(1998)).

Example 3 Screening for Compounds That Exhibit Viral Reaper ModulatingActivity

[0079] A cell culture of cells susceptible to viral Reaper-inducedeffects is established. The viral Reaper protein is administered to thecells (e.g., via transfection with a suitable plasmid and optionally areporter protein such as B-galactosidase; or by direct administration ofthe viral reaper protein to the cell). A test compound is alsoadministered to a test population of the cells. Administration of thetest compound may occur prior to, concurrently with, or afteradministration of the viral Reaper protein. The effect of administrationof the test compound on apoptosis of cells is compared to a controlpopulation of cells that did not receive the test compound. Significantdifferences in apoptosis (and/or caspase activation or other indicatorsof apoptosis) between the test and control cells indicates that the testcompound modulates viral reaper protein activity.

[0080] Alternatively, extracts prepared from Xenopus eggs are used asdescribed herein to test compounds for viral reaper-modulating effects.The test compound is included in an assay as described herein, andcaspase activity in cells receiving the test compound is compared tocontrol cells that do not receive the test compound. Significantdifferences in caspase activity production between the test and controlextracts indicates that the test compound modulates viral reaper proteinactivity.

Example 4 Binding of Drosophila and Viral Reaper to Scythe

[0081] Glutathione S-transferase, Drosophila reaper, and two viralreaper proteins were examined to detect the ability to bind to scytheprotein. It has previously been shown that drosophila reaper binds toXenopus scythe protein to induce apoptosis in Xenopus oocytes. Scythehas been indicated as an apoptotic regulator that is an essentialcomponent in reaper-induced apoptosis; immunodepletion of scythe fromXenopus egg extracts prevents reaper-induced apoptosis without affectingapoptosis triggered by activated caspase. (Thress et al., EMBO J17:6135-43 (1998); Evans et al, EMBO J 16:7372-81 (1997); Thress et al.,EMBO J 18:5486 (1999)).

[0082] Glutathione S-transferase (GST), a Drosophila Reaper-GSTconstruct (RPR), San Angelo virus reaper—GST construct (SA), andCalifornia Encephalitis virus reaper—GST construct (CE) were produced inBL21 bacterial strains (using standard techniques as are known in theart) and purified using glutathione sepharose beads. Two preparations ofGST prepared by two different individuals were utilized as controls(GST1 and GST2); two preparations of Drosophila reaper prepared by twoindividuals were also used (RPR1 and RPR2). The Xenopus crude eggextract was prepared as described in Evans et al., EMBO J, 16:7372(1997) and Thress et al., EMBO J, 17:6135 (1998).

[0083] The beads were incubated in Xenopus crude egg extract for anhour. The beads were then washed several times in egg lysis buffer (ELB;250 mM sucrose, 2.5 mM MgCl2, 1.0 mM dithiothreitol (DTT), 50 mM KCl, 10mM HEPES), pH 7.4, boiled, and run in an SDS-PAGE gel (using techniquesas are well-known in the art) and subjected to Western analysis (usingtechniques that are well known in the art) to detect binding of scytheto the beads. An aliquot of Xenopus crude egg extract (CS) without anyreaper or GST was also run on SDS-PAGE gel as a positive control.

[0084] As shown in FIG. 2, no binding was seen with either of the twoGST preparations. Drosophila reapers (RPR1 and RPR2) bound scythe; theXenopus crude egg extract (CS), which contains scythe, was included toshow the position of scythe. Additionally, both of the viral reapers (SAand CE) showed scythe binding. The reduced intensity of the Western blotcorresponding to viral CE reaper may indicate reduced binding to scythe(compared to the other reaper proteins tested) in this assay, as totalreaper or viral reaper protein was maintained constant.

Example 5 Activation of Caspase

[0085] Caspase activation was measured using three separate preparationsof Drosophila Reaper-GST, San Angelo virus reaper-GST (SA), andCalifornia Encephalitis virus reaper-GST(CE) (prepared and purified asdescribed above). GST was used as a control.

[0086] Cell-free extracts were prepared from Xenopus oocytes asdescribed in Thress et al., EMBO J, 17:6135 (1998), at page 6141. Whileit is known that these cell-free extracts will spontaneously releasemitochondrial cytochrome c and activate endogenous caspases afterprolonged incubation at room temperature, it has further been shown thatthe addition of drosophila Reaper measurably accelerates this process,triggering mitochondrial cytochrome c release, caspase activation, andfragmentation of added nuclei. (Newmeyer et al., Cell 79:353 (1994);Evans et al., EMBO J. 16:7372 (1997)).

[0087] Briefly to assess the ability of a reaper or viral reaper proteinto induce caspase release, and thus by inference induce apoptosis, 10 ulof GST-reaper or GST-viral reaper protein (released from GST beads withglutathione under standard conditions) at ˜0.5 to 1 mg/ml was mixed with100 ul Xenopus oocyte extract. This mixture was incubated for up to 7 hrand assayed for caspase activation a specific times. To measure thecaspase activity in the incubation mixtures, 3 μl of each incubationsample were mixed and incubated with 90 μl of assay buffer (50 mM HEPESpH 7.5, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% glycerol) andthe colorimetric substrate Ac-DEVD-pNA (final concentration 200 μM)[BioMol Caspase-3 assay system; BioMol Research Laboratories Inc.,Plymouth Meeting, Pennsylvania] at 37° C. At various time points,absorbance was measured at 405 nm; the measure of absorbance is directlyproportional to caspase activation.

[0088] As shown by FIG. 3 (Y-axis=absorbance at 405 nm; X-axis=time (inhours), the GST control did not result in activation of caspase, whileeach of the three Drosophila reaper preparations activated caspase,although the time at which activation was first detected varied amongthe reaper preparations. Both of the viral reaper preparations resultedin caspase activation. The SA virus reaper activated caspase over a timecourse similar to that of the Reaper 4/5 preparation in this assay. TheCE virus reaper resulted in initial caspase activation at the 6-7 hourtimepoint, however, the experiment did not extend beyond this timepoint.

1 34 1 65 PRT Drosophila melanogaster 1 Met Ala Val Ala Phe Tyr Ile ProAsp Gln Ala Thr Leu Leu Arg Glu 1 5 10 15 Ala Glu Gln Lys Glu Gln GlnIle Leu Arg Leu Arg Glu Ser Gln Trp 20 25 30 Arg Phe Leu Ala Thr Val ValLeu Glu Thr Leu Arg Gln Tyr Thr Ser 35 40 45 Cys His Pro Lys Thr Gly ArgLys Ser Gly Lys Tyr Arg Lys Pro Ser 50 55 60 Gln 65 2 92 PRT ArtificialSequence Description of Artificial Sequence Majority viral reapersequence 2 Met Met Ser His Pro Gln Val Gln Met Asp Leu Ile Leu Met GlnGly 1 5 10 15 Met Trp His Leu Val Leu Asn Met Gly Asn Leu Ser Ile CysGln Pro 20 25 30 Leu Gly Ser Ser Ser Leu Met Pro Gln Lys Pro Lys Leu LeuSer Leu 35 40 45 Val Ser Arg Arg Gly Lys Leu Ile Leu Asn Leu Glu Ser GlyArg Trp 50 55 60 Arg Leu Ser Ile Ile Ile Phe Leu Glu Thr Gly Thr Thr GlnLeu Val 65 70 75 80 Thr Thr Ile Leu Pro Ser Thr Gly Phe Gln Asp Ile 8590 3 97 PRT San Angelo virus 3 Met Met Ser His Gln Pro Val Gln Met AspLeu Ile Leu Met Gln Gly 1 5 10 15 Ile Trp His Ser Val Leu Asn Met GlySer Arg Ser Val Cys Leu Gln 20 25 30 Leu Gly Ser Ser Ser Ser Met Pro GlnLys Pro Lys Leu Leu Ser Arg 35 40 45 Val Asn Gln Arg Gly Lys Gln Ile LeuAsn Leu Ala Ser Gly Arg Trp 50 55 60 Arg Leu Ser Ile Ile Ile Phe Leu GluThr Gly Thr Ile Gln Leu Thr 65 70 75 80 Thr Ser Ile Leu Pro Ser Thr AspCys Leu Asp Thr Trp Leu Asp Gly 85 90 95 Phe 4 90 PRT Snowshoe harevirus 4 Met Met Ser His Gln Gln Val Gln Met Asp Leu Ile Leu Met Gln Gly1 5 10 15 Ile Trp His Ser Val Leu Asn Met Gln Asn Gln Ser Ile Leu LeuGln 20 25 30 Leu Gly Ser Ser Ser Ser Met Pro Arg Pro Arg Leu Leu Ser ArgVal 35 40 45 Ser Gln Arg Gly Arg Gln Ile Leu Asn Leu Glu Ser Gly Arg TrpArg 50 55 60 Leu Ser Ile Ile Ile Phe Leu Glu Thr Gly Thr Ile Gln Leu ThrThr 65 70 75 80 Ile Leu Pro Ser Thr Asp Cys Gln Asp Ile 85 90 5 92 PRTLa Crosse virus 5 Met Met Ser His Gln Gln Val Gln Met Asp Leu Ile LeuMet Gln Gly 1 5 10 15 Ile Trp Thr Ser Val Leu Lys Met Gln Asn His SerThr Leu Leu Gln 20 25 30 Leu Gly Ser Ser Ser Ser Met Leu Gln Arg Pro ArgLeu Leu Ser Arg 35 40 45 Val Ser Gln Arg Gly Arg Leu Thr Leu Asn Leu GluSer Gly Arg Trp 50 55 60 Arg Leu Ser Ile Ile Ile Phe Leu Glu Thr Gly ThrThr Gln Leu Val 65 70 75 80 Thr Thr Ile Leu Pro Ser Thr Asp Tyr Leu GlyIle 85 90 6 97 PRT California encephalitis virus 6 Met Met Ser His ProGln Val Gln Met Asp Leu Ile Leu Met Gln Gly 1 5 10 15 Met Trp Thr SerVal Leu Asn Met Gly Asn Gln Leu Thr Leu Leu Gln 20 25 30 Leu Gly Ser SerSer Ser Met Pro Gln Arg Pro Arg Leu Leu Ser Arg 35 40 45 Val Ser Gln ArgGly Lys Leu Ile Leu Asn Leu Ala Ser Gly Arg Trp 50 55 60 Arg Leu Ser IleIle Ile Gly Gln Gln Thr Gly Thr Ile Gln Leu Val 65 70 75 80 Thr Thr IleLeu Pro Ser Thr Ala Ser Gln Asp Thr Leu Pro Asp Gly 85 90 95 Ser 7 92PRT Jerry Slough virus 7 Met Met Ser His Pro Gln Val Gln Met Asp Leu IleGln Met Gln Gly 1 5 10 15 Leu Trp His Leu Trp Leu Thr Thr Glu Ser LeuSer Ile Cys Gln Pro 20 25 30 Leu Gly Ser Ser Ser Leu Met Gln Gln Lys ProLys Leu Leu Ser Leu 35 40 45 Val Asn Arg Ser Gly Lys Leu Leu Leu Ser LeuGlu Ser Gly Arg Trp 50 55 60 Arg Ser Ser Ile Ile Ile Phe Leu Glu Thr GlyThr Thr Gln Leu Val 65 70 75 80 Thr Thr Ile Leu Pro Ser Ile Gly Phe GlnAsp Ile 85 90 8 92 PRT Jamestown Canyon virus 8 Met Met Ser His Pro GlnVal Gln Met Asp Leu Ile Gln Met Gln Gly 1 5 10 15 Leu Trp His Leu TrpLeu Thr Thr Glu Ser Leu Ser Ile Cys Gln Pro 20 25 30 Leu Gly Ser Ser SerLeu Met Gln Gln Lys Pro Lys Leu Leu Ser Leu 35 40 45 Val Asn Arg Ser GlyLys Leu Leu Leu Ser Leu Glu Ser Gly Arg Trp 50 55 60 Arg Ser Ser Ile IleIle Phe Leu Glu Thr Gly Thr Thr Gln Leu Val 65 70 75 80 Thr Thr Ile LeuPro Ser Ile Gly Phe Gln Asp Ile 85 90 9 92 PRT Keystone virus 9 Met MetSer His Pro Gln Val Gln Met Asp Leu Ile Leu Met Gln Gly 1 5 10 15 MetTrp His Leu Trp Leu Thr Met Gly Ser Arg Ser Val Cys Gln Pro 20 25 30 LeuGly Ser Ser Ser Leu Met Pro Gln Lys Pro Lys Leu Leu Ser Leu 35 40 45 ValSer Arg Ser Gly Arg Leu His Leu Ser Leu Glu Ser Gly Arg Trp 50 55 60 ArgSer Ser Ile Ile Ile Phe Leu Glu Thr Gly Thr Thr Gln Leu Val 65 70 75 80Thr Thr Ile Leu Pro Cys Thr Gly Phe Gln Asp Ile 85 90 10 97 PRT Melaovirus 10 Met Met Ser His Gln Gln Val Gln Met Asp Leu Ile Gln Met Gln Gly1 5 10 15 Ile Trp His Leu Gln Leu Arg Met Gly Lys Leu Ser Ile Cys GlnPro 20 25 30 Leu Gly Ser Ser Ser Leu Met Pro Gln Lys Pro Lys Leu Leu SerLeu 35 40 45 Val Asn Arg Arg Gly Lys Leu Leu Leu Asn Leu Glu Thr Gly ArgTrp 50 55 60 Lys Leu Ser Thr Ile Ile Phe Leu Glu Thr Gly Thr Thr Gln LeuVal 65 70 75 80 Thr Thr Ile Leu Pro Ser Ile Gly Phe Gln Asp Ile Leu ProAsp Gly 85 90 95 Cys 11 97 PRT Trivittatus virus 11 Met Met Leu His GlnGln Val Gln Thr Asp Leu Ile Pro Met Gln Gly 1 5 10 15 Met Trp His LeuLeu Leu His Met Pro Asp Arg Thr Ile Phe Leu Leu 20 25 30 Leu Gly Ser SerSer Ser Met Leu Pro Arg Pro Arg Met Leu Ser Arg 35 40 45 Glu Asn Gln ArgGly Arg Leu Val Leu Asn Leu Ala Ser Gly Arg Trp 50 55 60 Arg Trp Ser IleIle Ile Phe Leu Ala Thr Gly Thr Ile Gln Leu Val 65 70 75 80 Thr Thr IleLeu Pro Ser Thr Glu Phe Gln Ala Ile Ser Gln Asp Gly 85 90 95 Phe 12 97PRT Morro Bay virus 12 Met Met Ser His Pro Gln Val Gln Met Asp Leu IleLeu Met Gln Gly 1 5 10 15 Met Trp Thr Ser Val Leu Asn Met Gly Asn ArgLeu Thr Leu Leu Gln 20 25 30 Leu Gly Ser Ser Ser Ser Met Pro Gln Lys ProArg Leu Leu Ser Arg 35 40 45 Val Ser Gln Arg Gly Lys Leu Ile Leu Asn LeuAla Ser Gly Arg Trp 50 55 60 Arg Leu Ser Ile Ile Ile Phe Gln Gln Thr GlyThr Ile Gln Leu Val 65 70 75 80 Thr Thr Ile Leu Pro Ser Thr Ala Ser GlnAsp Thr Leu Pro Asp Gly 85 90 95 Ser 13 92 PRT Inkoo virus 13 Met MetSer His Pro Gln Val Gln Met Asp Leu Ile Gln Met Gln Gly 1 5 10 15 LeuTrp His Leu Trp Leu Thr Met Glu Asn Leu Leu Ile Trp Gln Pro 20 25 30 LeuGly Ser Ser Ser Leu Met Gln Gln Lys Pro Lys Leu Leu Ser Leu 35 40 45 ValAsn Arg Ser Gly Lys Leu Leu Leu Asn Leu Glu Ser Gly Arg Trp 50 55 60 ArgLeu Ser Ile Ile Ile Phe Leu Glu Thr Gly Thr Thr Gln Leu Val 65 70 75 80Thr Thr Ile Leu Pro Ser Thr Gly Phe Leu Asp Thr 85 90 14 92 PRT Serra doNavio virus 14 Met Met Ser His Gln Pro Val Gln Met Asp Leu Ile Gln MetGln Gly 1 5 10 15 Leu Trp His Leu Trp Leu Val Met Gly Ser Arg Ser IleLeu Gln Pro 20 25 30 Leu Glu Ser Ser Ser Leu Met Pro Gln Lys Pro Lys LeuLeu Ser Leu 35 40 45 Ala Ser Arg Arg Gly Lys Leu Leu Leu Ser Leu Glu ThrGly Arg Trp 50 55 60 Arg Leu Ser Ile Ile Ile Phe Leu Glu Thr Gly Thr ThrGln Leu Val 65 70 75 80 Thr Thr Ile Leu Pro Ser Thr Glu Phe Gln Asp Ile85 90 15 92 PRT South River virus 15 Met Met Ser His Pro Gln Val Gln MetAsp Leu Ile Gln Met Gln Gly 1 5 10 15 Leu Trp His Leu Trp Leu Thr MetGlu Asn Leu Ser Ile Cys Gln Pro 20 25 30 Leu Gly Ser Ser Ser Leu Met GlnGln Lys Pro Lys Leu Leu Ser Leu 35 40 45 Val Asn Arg Ser Gly Arg Leu IleLeu Asn Leu Glu Ser Gly Arg Trp 50 55 60 Arg Leu Ser Ile Ile Ile Phe LeuGlu Thr Gly Thr Thr Gln Leu Val 65 70 75 80 Thr Thr Ile Leu Pro Ser ThrGly Phe Leu Asp Ile 85 90 16 97 PRT Lumbo virus 16 Met Met Ser His ProPro Val Gln Met Asp Leu Ile Leu Met Gln Gly 1 5 10 15 Met Trp Thr PheVal Leu Asn Met Glu Asn Gln Ser Ile Ser Ile Pro 20 25 30 Leu Gly Ser PheSer Leu Met Pro Leu Arg Pro Arg Leu Leu Ser Leu 35 40 45 Val Ser Arg ArgGly Arg Leu Val Leu Asn Leu Glu Ser Gly Arg Trp 50 55 60 Arg Ser Ser IleIle Ile Phe Leu Glu Thr Gly Thr Thr Gln Leu Ile 65 70 75 80 Thr Thr IleLeu Pro Ser Thr Gly Cys Gln Gly Ile Trp Leu Asp Gly 85 90 95 Cys 17 97PRT Tahyna virus 17 Met Met Ser His Pro Pro Val Gln Met Asp Leu Ile LeuMet Gln Gly 1 5 10 15 Met Trp Thr Ser Val Leu Asn Met Gly Lys Gln LeuIle Ser Ile Pro 20 25 30 Leu Gly Ser Ser Ser Leu Met Pro Gln Lys Pro LysLeu Leu Ser Leu 35 40 45 Val Ser Arg Arg Gly Arg Leu Val Leu Asn Leu GluSer Gly Arg Trp 50 55 60 Arg Ser Ser Ile Ile Ile Phe Leu Glu Thr Gly ThrThr Gln Leu Ile 65 70 75 80 Thr Thr Ile Leu Pro Ser Thr Gly Cys Thr GlyIle Trp Leu Asp Gly 85 90 95 Cys 18 23 PRT Artificial SequenceDescription of Artificial Sequence portion of Drosophila melanogasterGrim protein 18 Met Ala Ile Ala Phe Tyr Ile Pro Asp Gln Ala Gln Leu LeuAla Arg 1 5 10 15 Ser Tyr Gln Gln Asn Gly Gln 20 19 198 DNA Drosophilamelanogaster 19 atggcagtgg cattctacat acccgatcag gcgactctgt tgcgggaggcggagcagaag 60 gagcagcaga ttctccgctt gcgggagtca cagtggagat tcctggccaccgtcgtcctg 120 gaaaccctgc gccagtacac ttcatgtcat ccgaagaccg gaagaaagtccggcaaatat 180 gcgaagccat cgcaatga 198 20 294 DNA San Angelo virus 20atgatgtcgc atcaaccggt gcaaatggat ttgatcctga tgcagggtat ctggcattct 60gtgttaaaca tggggagtcg atcagtttgt cttcagttag gatcttcttc ctcaatgccg 120caaaagccaa agctgctctc tcgcgtaaac cagagaggaa agcaaatcct aaatttggcg 180agtggcaggt ggagattgtc aataatcatt ttcctggaaa caggaacaat ccaattgaca 240acctcgatct taccatccac agattgtctg gatacctggc tagatgggtt ctag 294 21 279DNA Snowshoe hare virus 21 atgatgtcgc atcaacaggt gcaaatggat ttgatcctgatgcagggtat atggcattct 60 gtgttaaata tgcagaatca gtcaatcttg ctgcagttaggatcttcttc ctcaatgccg 120 caaaggccaa ggctgctctc tcgcgtaagc cagagaggaaggcaaatcct aaatttggag 180 agtggcaggt ggaggttgtc aataatcatt ttcctggaaacaggaacaat ccaattaaca 240 gcgacgatct taccatccac agattgtcag gatatttag 27922 279 DNA LaCrosse Virus 22 atgatgtcgc atcaacaggt gcaaatggat ttgatcctgatgcagggtat atggacttct 60 gtgttaaaaa tgcagaatca ctcaaccttg ctgcagttaggatcttcttc ctcaatgctg 120 caaaggccaa ggctgctctc tcgcgtaagc cagagaggaaggctaaccct aaatttggag 180 agtggcaggt ggaggttatc aataatcatt ttcctggaaacaggaacaac ccaattggta 240 acaacgatct taccatccac agattatctg ggtatttag 27923 294 DNA California encephalitis virus 23 atgatgtcgc atccacaggtgcaaatggat ttgatcctga tgcagggtat gtggacttct 60 gtgctaaaca tggggaatcaattaaccttg ctgcagttag gatcttcttc ctcaatgccg 120 caaaggccaa ggctgctctctcgcgtaagc cagagaggaa agctaatcct aaatttggcg 180 agtggcaggt ggaggttgtcaataatcatt ttccagcaaa caggaacaat ccaattggta 240 acaacgatct taccatccaccgcatctcag gataccttgc cagatgggtc ctag 294 24 279 DNA Jerry Slough virus24 atgatgtcgc atccacaggt gcaaatggat ttgatccaga tgcagggttt gtggcattta 60tggctgacca cggagagtct atcaatctgt cagccgttag gatcttcttc cttaatgcag 120caaaagccaa agctgctctc gctcgtaaac cggagcggaa agctactcct aagtttggag 180agtggcaggt ggagatcatc aataatcatt ttcctggaaa caggaacaac ccaattggta 240acaacgatct taccatccat aggctttcag gatatctag 279 25 279 DNA JamestownCanyon virus 25 atgatgtcgc atccacaggt gcaaatggat ttgatccaga tgcagggtttgtggcattta 60 tggctgacca cggagagtct atcaatctgt cagccgttag gatcttcttccttaatgcag 120 caaaagccaa agctgctctc gctcgtaaac cggagcggaa agctactcctaagtttggag 180 agtggcaggt ggagatcgtc aataatcatt tttctggaaa caggaacaacccaattggta 240 acaacgatct taccatccat aggctttcag gatatctag 279 26 279 DNAKeystone virus 26 atgatgtcgc atccacaggt gcaaatggat ttgatcctga tgcagggtatgtggcattta 60 tggctaacca tggggagtcg atcagtctgt caaccgttag gatcttcttccttaatgccg 120 caaaagccaa agctgctctc actcgtaagc cggagcggaa ggctacacctaagtttggag 180 agtggcaggt ggagatcgtc aataatcatt ttcctggaaa caggaacaacccaattggta 240 acaacgatct taccttgcac cggatttcag gatatctag 279 27 294 DNAMelao virus 27 atgatgtcgc atcaacaggt gcaaatggat ttgatccaga tgcagggtatctggcattta 60 caattgcgca tggggaagct atcaatttgt cagccgttag gatcttcttccttaatgccg 120 caaaagccaa agctgctctc tctcgtaaac cggagaggaa agctactcctaaatttggag 180 actggcaggt ggaaattgtc aacaatcatt ttcctggaaa caggaacaacccaattggta 240 acaacgatct taccatccat cggctttcag gatatcttgc cagatgggtgctag 294 28 294 DNA Trivittatus virus 28 atgatgctcc atcaacaggtgcaaacggat ttgatcccga tgcagggtat gtggcattta 60 ttgctgcaca tgccggatcgtacgatcttt ctgctgttag gatcttcttc ctcaatgctg 120 ccaaggccaa gaatgctctctcgagaaaac cagaggggaa ggttagtatt aaatttggcg 180 agtggtcggt ggaggtggtcaataatcatt ttcctggcaa caggaacaat ccaattggta 240 acaacgatct taccatccacagaatttcag gctatctcgc aagatgggtt ctag 294 29 294 DNA Morro Bay virus 29atgatgtcgc atccacaggt gcaaatggat ttgatcctga tgcagggtat gtggacttct 60gtgctaaaca tggggaatcg attaaccttg ctgcagttag gatcttcttc ctcaatgccg 120caaaagccaa ggctgctctc tcgcgtaagc cagagaggaa agctaatcct aaatttggcg 180agtggcaggt ggagattgtc aataatcatt ttccagcaaa caggaacaat ccaattggta 240acaacgatct taccatccac cgcatctcag gataccttgc cagatgggtc ctag 294 30 279DNA Inkoo virus 30 atgatgtcgc atccacaggt gcaaatggat ttgatccagatgcagggttt gtggcattta 60 tggctgacca tggagaatct attaatttgg cagccgttaggatcttcttc cttaatgcag 120 caaaagccaa agctgctctc gctcgtaaac cggagcggaaagctactcct aaatttggag 180 agtggcaggt ggagattgtc aataatcatt ttcctggaaacaggaacaac ccaattggta 240 acaacgatct taccatccac cggctttctg gatacttag 27931 279 DNA Serra do Navio virus 31 atgatgtcgc atcaaccggt gcaaatggatttgatccaga tgcagggttt gtggcattta 60 tggctggtca tggggagtcg atcaatcttacagccgttag aatcttcttc cttaatgccg 120 caaaagccaa agctgctctc tctcgcaagccggagaggaa agctactcct aagtttggag 180 actggcaggt ggagattgtc aataatcattttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccatccac agaatttcaggatatttag 279 32 279 DNA South River virus 32 atgatgtcgc atccacaggtgcaaatggat ttgatccaga tgcagggttt gtggcattta 60 tggctgacca tggagaatctatcaatctgt cagccgttag gatcttcttc cttaatgcag 120 caaaagccaa agctgctctcgctcgtaaac cggagcggaa ggctaatcct aaatttggag 180 agtggcaggt ggagattgtcaataatcatt ttcctggaaa caggaacaac ccaattggta 240 acaacgatct taccatccaccggctttctg gatatttag 279 33 294 DNA Lumbo virus 33 atgatgtcgc atccaccggtgcaaatggat ttgatcctga tgcagggtat gtggactttt 60 gtgttaaaca tggagaatcaatcaatctcc attccgttag gatctttttc cttaatgccg 120 ctaaggccaa ggctgctctcgctcgtaagc cggagaggaa ggctagtcct aaatttggag 180 agtggcaggt ggagatcgtcaataatcatt ttcctggaaa caggaacaac ccaattgata 240 acaacgatct taccatccaccggctgtcag ggtatctggc tagatgggtg ttag 294 34 294 DNA Tahyna virus 34atgatgtcgc atccaccggt gcaaatggat ttgatcctga tgcagggtat gtggacttct 60gtattaaaca tggggaagca attaatctcc attccgttag gatcttcttc cttaatgccg 120caaaagccaa agctgctctc gctcgtaagc cggagaggaa ggctagtcct aaatttggag 180agtggcaggt ggaggtcgtc aattatcatt ttcctggaaa caggaacaac ccaattgata 240acaacgatct taccatccac cggctgtacg ggtatttggc tagatgggtg ctag 294

That which is claimed is:
 1. (amended) A method of screening a compoundto determine whether the compound affects caspase activation induced bya viral reaper protein, comprising: a) obtaining a vertebrate cellextract in which the addition of isolated viral reaper protein inducesdetectable caspase activation; b) adding isolated viral reaper proteinand a test compound to said vertebrate cell extract; c) measuringcaspase activation; and d) comparing caspase activation that occurs inthe presence of the test compound to that which would be expected in theabsence of the test compound; where a decrease in caspase activationcompared to that which would be expected in the absence of the testcompound indicates that said compound inhibits viral reaper-inducedcaspase activation, and an increase in caspase activation compared tothat which would be expected in the absence of the test compoundindicates that said test compound enhances viral reaper-induced caspaseactivation.
 2. (amended) A method according to claim 1 where saidisolated viral reaper protein comprises an amino acid sequence selectedfrom SEQ ID NOs: 2-17.
 3. (amended) A method according to claim 1 wheresaid isolated viral reaper protein is from a virus of the FamilyBunyaviridac.
 4. A method according to claim 1 where said vertebratecell extract is obtained from Xenopus oocytes.
 5. (amended) A method ofscreening a compound to determine whether the compound affects apoptosisinduced by a viral reaper protein, comprising: a) obtaining a populationof cells, the cells of a type in which the addition of isolated viralreaper protein induces apoptosis; b) administering isolated viral reaperprotein and a test compound to said cells; c) measuring apoptosis thatoccurs in said cell population; and d) comparing apoptosis that occursin the presence of the test compound to that which would be be expectedin the absence of the test compound; where a decrease in apoptosisindicates that said compound inhibits viral reaper-induced apoptosis,and an increase in apoptosis compared to that which would be expected inthe absence of the test compound indicates that said test compoundenhances viral reaper-induced apoptosis.
 6. A method according to claim5 wherein said population of cells consists of vertebrate cells. 7.(amended) A method according to claim 5 where said isolated viral reaperprotein comprises an amino acid sequence selected from SEQ ID NOs: 2-17.8. (amended) A method according to claim 5 where said isolated viralreaper protein is from a virus of the Family Bunyaviridae.
 9. (amended)A method according to claim 5 wherein said isolated viral reaper proteinis administered by transfecting cells with a nucleotide moleculeencoding the viral reaper protein.
 10. A method according to claim 5where apoptosis is measured by a criterion selected from the groupconsisting of caspase activation, cell death, or cellular DNAdegradation over time.
 11. (amended) A method of screening a compoundfor viral reaper modulating activity, comprising: (a) obtaining apopulation of cells modified to express an isolated viral reaperprotein; (b) administering to a test population of said cells a testcompound; and (c) comparing apoptosis occurring in said test populationof cells to that which would be expected in a population of cells thatdid not receive said test compound; where a reduction in apoptosisindicates that said test compound inhibits viral reaper-inducedapoptosis, and an increase in apoptosis indicates said test compoundenhances viral-reaper induced apoptosis.
 12. A method according to claim11 where said population of cells consists of insect cells.
 13. A methodaccording to claim 11 where said population of cells consists ofvertebrate cells.
 14. A method according to claim 11 where apoptosis ismeasured by a criterion selected from caspase activation, cell death, orcellular DNA degradation over time.
 15. (amended) A method according toclaim 11 where said isolated viral reaper protein comprises an aminoacid sequence selected from SEQ ID NOs: 2-17.
 16. (amended) A methodaccording to claim 11 where said isolated viral reaper protein is from avirus of the Family Bunyaviridac.
 17. A method of inducing apoptosis ina vertebrate cell by administering an isolated viral reaper protein tosaid cell, in an amount sufficient to enhance apoptosis over that whichwould be seen in the absence of viral reaper protein.
 18. A methodaccording to claim 17 wherein said vertebrate cell is a mammalian cell.19. A method according to claim 17 where administration of the viralreaper protein is by transfection with an isolated nucleic acid moleculeencoding said viral reaper protein.
 20. A method according to claim 17where an expression vector containing an isolated nucleotide sequenceencoding a viral reaper protein is administered to said cell.